Enamel matrix proteins, present in the enamel matrix, are most well-known as precursors to enamel. Prior to cementum formation, enamel matrix proteins are deposited on the root surface at the apical end of the developing tooth-root. There is evidence that the deposited enamel matrix is the initiating factor for the formation of cementum. Again, the formation of cementum in itself is associated with the development of the periodontal ligament and the alveolar bone. Enamel matrix proteins can therefore promote periodontal regeneration through mimicking the natural attachment development in the tooth (Gestrelius S, Lyngstadaas S P, Hammarstrøm L. Emdogain—periodontal regeneration based on biomimicry. Clin Oral Invest 4:120-125 (2000)).
Isolated enamel matrix proteins are able to induce not only one but an orchestrated cascade of factors, naturally found in tissues developing adjacent to the enamel matrix They mimic the natural environment of a developing tissue and thus mimic a natural stimulation for tissue regeneration, cell differentiation and/or maturation.
Enamel matrix derivative (EMD), in the form of a purified acid extract of proteins from pig enamel matrix, has previously been successfully employed to restore functional periodontal ligament, cementum and alveolar bone in patients with severe tooth attachment loss (Hammarström et al., 1997, Journal of Clinical Periodontology 24, 658-668).
Furthermore, in studies on cultured periodontal ligament cells (PDL), it was shown that the attachment rate, growth and metabolism of these cells were significantly increased when EMD was present in the cultures. Also, cells exposed to EMD showed increased intracellular cAMP signalling and autocrine production of growth factors, when compared to controls. Epithelial cells on the other hand, although increasing cAMP signalling and growth factor secretion when EMD was present, were inhibited in both proliferation and growth (Lyngstadaas et al., 2001, Journal of Clinical Periodontology 28, 181-188).
Enamel matrix proteins and enamel matrix derivatives (EMD) have previously been described in the patent literature to be able to induce hard tissue formation (i.e. enamel formation, U.S. Pat. No. 4,672,032 (Slavkin)), endorse binding between hard tissues (EP-B-0 337 967 and EP-B-0 263 086), promote open wound healing, such as of skin and mucosa, have a beneficial effect on treatment of infections and inflammatory diseases (EP-1059934 and EP-01201915.4), induce regeneration of dentin (WO 01/97834), promote the take of a graft (WO 00/53197), induce apoptosis in the treatment of neoplasms (WO 00/53196), regulate imbalance in an immune response to a systemic infection or inflammation (WO 03/024479), and to facilitate filling a wound cavity and/or tissue defect following from a procedure and/or trauma, such as a cytoreductive surgery (WO 02/080994).
The enamel matrix is composed of a number of proteins, such as amelogenins, enamelin, tuft protein, proteases, and albumin. Amelogenins, a major constituent of the enamel matrix, are a family of hydrophobic proteins derivable from a single gene by alternative splicing and controlled post secretory processing. They are highly conserved throughout vertebrate evolution and demonstrate a high overall level of sequence homology among all higher vertebrates examined (80%). In fact, the sequences of porcine and human amelogenin gene transcript differ only in 4% of the bases (i.e. they are approximately 90-96% identical). Thus, enamel matrix proteins, although of porcine origin, are considered “self” when encountered in the human body and can promote dental regeneration in humans without triggering allergic responses or other undesirable reactions. Nonetheless, the plurality of structures identified in the different amelogenins studied, that will even occur in the same individual animal or human dentitions, clearly gives rise to speculations on the extreme specificity of the structures that work in concert in a “normal” amelogenesis. E.g., a single base mutation in the x-chromosomal amelogenin gene, which results in a single proline to threonine change in the expressed human amelogenin, does give rise to amelogenesis imperfecta.
During cementogenesis in the developing tooth, amelogenin degrades into smaller pieces, and these pieces seem to interact differentially with the surrounding tissue and promote serial steps in the development of the periodontal system. As already described in Fincham et al, 1993, enamel contains a complex of amelogenin proteins which includes components ranging in size from 5-25 kDa. This is due to the expression and secretion of a family of amelogenins derivable from multiple mRNAs generated by differential splicing from one or two copies of the amelogenin gene, located on the X and Y chromosome. What is more, subsequent to secretion, these proteins appear further to undergo extensive proteolytic processing. Because of this extensive alternative splicing of the primary transcript and the following proteolytic processing of the secreted proteins, it has been difficult to assign functions to individual amelogenins. The pattern of splicing is unique for each amelogenin gene yet investigated, even when two copies of the gene are expressed in the same cell. Despite the high conservation of amelogenin sequences across species, diversity in the pattern of RNA splicing thus leads to significant differences in the number and character of amelogenin isoforms in the developing enamel matrix.
The need for a more refined use of enamel matrix protein, e.g. to induce specific steps during periodontal development, such as de novo bone formation or cementogenesis, or to mimic them in medical treatments has long been felt in the field. The need for an efficient possibility to synthesize single defined polypeptide sequences for use as separate and/or combined active components for inducing a specific desired effect has long been sought for, but, due to the complexity of the endogenous expression and processing of amelogenin proteins, attempts at separation of certain closely defined fractions and/or polypeptides or fragments of polypeptides from e.g. porcine tissues with specific biological activities have meet with severe obstacles.
To date, two classes of amelogenin proteins have been described in the size of between 5-6 kDa, namely leucine-rich amelogenin polypeptide (LRAP) and tyrosine-rich amelogenin polypeptide (TRAP). LRAP is translated from a shorter mRNA that has the coding regions from exons 4, 5 and part of 6 deleted during splicing. Due to its potential important regulatory effect as one of the processed fragments found of amelogenin, it was 2004 investigated by Boabaid et al, (Boabaid F., et al, J. Periodontol, Vol 75, No. 8, 2004) but was reported not to have any effect on cell proliferation in itself. What is more, it decreased the number of cementoblasts in cell culture, contrary to EMD which promotes cell proliferation of cementoblasts in vitro and full length amelogenin, which has no reported effect.
Two human tyrosine-rich amelogenin polypeptides (TRAPs) of approximately 5 kDa in size have prior been identified (see Fincham et al., 1989). These polypeptides were found to be of 42 (TRAP-2) and 44 (TRAP-1) amino acid residues in length; two forms of TRAP molecules, differing only by cleavage of a carboxy-terminal dipeptide, which were described to be a general feature of human and other mammalian enamel proteins, probably being derived by postsecretory cleavage from the primary extracellular amelogenin. No specific biological effect has so far been attributed to these polypeptides either. In WO 2009/157869, the present inventors finally were able to describe 2 naturally occurring porcine N-terminal amelogenin polypeptide fragments that together were shown to be able to induce osteogenic activity, such as proliferation of precursor cells and early differentiation of osteoblasts (TRAP 43 and TRAP 45).
The present invention now for the first time discloses all components of a newly identified low molecular weight fraction of isolated enamel matrix derivatives that can clearly be shown to be more effective in reducing the inflammatory response in a targeted soft tissue than the complete enamel matrix extract (EMD). In particular, one specific polypeptide is identified that has been prior unknown and which stimulates the tissue formation phase of the wound healing process. Its use is herein described in promoting and/or improving soft tissue regeneration and/or stimulation of angiogenesis, such as in periodontal tissues.